P340: IN VITRO AND IN VIVO EFFICACY OF A NOVEL KINASE INHIBITOR TARGETING JAK2 GENE REARRANGEMENTS IN CHILDHOOD ACUTE LYMPHOBLASTIC LEUKEMIA
Authors: M. Quadri, C. Saitta, S. Palamini, C. Palmi, A. Biondi, G. Cazzaniga, G. Fazio
Journal: HemaSphere (2022)

Background: Although risk-based treatment is curative for 85% of children with B-cell precursor acute lymphoblasticleukemia (BCP-ALL), relapse remains a leading cause of mortality, urging the need of novel molecular targets. The JAK/STAT alterations represent about 7% of the ‘Philadelphia-like’ cases. JAK2 gene encodes for a non-receptor tyrosine kinase fundamental for hematopoiesis and itsmutations have been widely studied, whereas JAK2 fusion genes are still poorly characterized. Aims: This study aims to identify JAK2 fusion genes among BCP-ALL pediatric patients, developing a target strategy in preclinical models. Methods: We applied RNA Next Generation Sequencing to find JAK2 fusion genes in a cohort of high risk BCP-ALL pediatric patients. Fusions were validated by RT-PCR and/or FISH. In vivo expansion of patients’ cells has been carried out in NSG mice. After drug treatments with JAK2 inhibitors, phosphoflow and apoptosis assays were done. Results: We identified 10 pediatric cases carrying a JAK2 fusion with different partners, where PAX5 gene was the only recurrent.After in vivo expansion of cells from 3 cases, carrying PAX5::JAK2, ATF7IP::JAK2 and ZEB2::JAK2, we demonstrated that JAK2 signaling was activeat basal level, through phosphorylation of JAK2 Y1007-1008 compared to cases wild type for JAK2 and CRLF2 (+70%, two-tailed P value 0.03); and also compared to P2RY8::CRLF2 rearrangements and JAK2 mutation(+40%, two-tailed P value 0.16). The JAK2 downstream effectors pS727-STAT3 and pY694-STAT5 were also activated. We targeted JAK2using CHZ868, a new class-II tyrosine kinase inhibitor (TKI) (Novartis, Basel, CH). After 30 minutes and till48h, we appreciated a mean inhibition of -62% of Y1007-1008 JAK2 residues in PAX5::JAK2, -22% in ATF7IP::JAK2 and -35% in ZEB2::JAK2. Contemporarily, we observed a decrease of pS727-STAT3 (-35-50%) and pY694-STAT5 (-15-50%) and the significant reduction of phosphorylation on PI3K pathway,downregulating PDPK1, AKT, 4pEBP1 and pS6. After 48h monotherapy treatment by CHZ868, we detected decrease in cell viability (20-75% at IC50). In combination with dexamethasone, a further decrease of viability was observed. In the PAX5::JAK2 case, we also performed treatments with BIBF1120/Nintedanib, LCK inhibitor (activated downstream to PAX5 fusions) and we observed a 20% reduction of cell viability. Importantly, combination of BIBF1120 and CHZ868 showed a synergistic effect (-45%, at IC50). Moreover, ruxolitinib caused autophagy as observed by higher levels of LC3-II compared to untreated cells (+45%, p<0.01), with reduction of apoptosis. Indeed, active caspase 3 increased after ruxolitinib and chloroquine (autophagy inhibitor) combination(+20% vs ruxolitinib alone, p<0.01). Instead, CHZ868 alone or in combination with chloroquine does not induce autophagy, with no effect on bothLC3-II and active caspase 3 levels. Finally, we demonstrated the in vivo efficacy of CHZ868 in PAX5::JAK2, ATF7IP::JAK2 and ZEB2::JAK2 patient-derived-xenografts. After two weeks of 30mg/Kg daily treatment of CHZ868, we observed a significant reduction of leukemic CD10+/CD19+ cells both in BM(-43-85%), spleen(-72-89%), CNS (-13-62%) and PB (-46-80%).Moreover, CHZ868 in vivo treatment significantly reduced the phosphorylation of pJAK2 (-18-46%), pSTAT5 (-23-71%) and pAKT(Ser473) (-18-34%). Summary/Conclusion: CHZ868 is a promising drugfor the treatment of JAK2 fusionsBCP-ALL. Further studies will include combination with standard chemotherapy drugs, by reducing the intensity and toxicity of chemotherapy.